Kinetic variations determine the product pattern of phytoene desaturase from Rubrivivax gelatinosus

In bacteria and fungi, the degree of carotenoid desaturation is determined by a single enzyme, the CrtI-type phytoene desaturase. In different organisms, this enzyme can carry out either three, four or even five desaturation steps. The purple bacterium Rubrivivax gelatinosus is the only known species in which reaction products of a 3-step and a 4-step desaturation (i.e. neurosporene and lycopene derivatives) accumulate simultaneously. The properties of this phytoene desaturation to catalyze neurosporene or lycopene were analyzed by heterologous complementations in Escherichia coli and by in vitro studies. They demonstrated that high enzyme concentrations or low phytoene supply favor the formation of lycopene. Under these conditions, CrtI from Rhodobacter spheroides can be forced in vitro to lycopene formation although this carotene is not synthesized in this species. All results can be explained by a model based on the competition between phytoene and neurosporene for the substrate binding site of phytoene desaturase. Mutations in CrtI from Rvi. gelatinosus have been generated resulting in increased lycopene formation in Escherichia coli. This modification in catalysis is due to increased amounts of CrtI protein.     

Cooperation of two carotene desaturases in the production of lycopene in Myxococcus xanthus

In Myxococcus xanthus, all known carotenogenic genes are grouped together in the gene cluster carB-carA, except for one, crtIb (previously named carC). We show here that the first three genes of the carB operon, crtE, crtIa, and crtB, encode a geranygeranyl synthase, a phytoene desaturase, and a phytoene synthase, respectively. We demonstrate also that CrtIa possesses cis-to-trans isomerase activity, and is able to dehydrogenate phytoene, producing phytofluene and zeta-carotene. Unlike the majority of CrtI-type phytoene desaturases, CrtIa is unable to perform the four dehydrogenation events involved in converting phytoene to lycopene. CrtIb, on the other hand, is incapable of dehydrogenating phytoene and lacks cis-to-trans isomerase activity. However, the presence of both CrtIa and CrtIb allows the completion of the four desaturation steps that convert phytoene to lycopene. Therefore, we report a unique mechanism where two distinct CrtI-type desaturases cooperate to carry out the four desaturation steps required for lycopene formation. In addition, we show that there is a difference in substrate recognition between the two desaturases; CrtIa dehydrogenates carotenes in the cis conformation, whereas CrtIb dehydrogenates carotenes in the trans conformation.     

Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation.

Carotenoids are C40 tetraterpenoids synthesized by nuclear-encoded multienzyme complexes located in the plastids of higher plants. To understand further the components and mechanisms involved in carotenoid synthesis, we screened Arabidopsis for mutations that disrupt this pathway and cause accumulation of biosynthetic intermediates. Here, we report the identification and characterization of two nonallelic albino mutations, pds1 and pds2 (for phytoene desaturation), that are disrupted in phytoene desaturation and as a result accumulate phytoene, the first C40 compound of the pathway. Surprisingly, neither mutation maps to the locus encoding the phytoene desaturase enzyme, indicating that the products of at least three loci are required for phytoene desaturation in higher plants. Because phytoene desaturase catalyzes an oxidation reaction, it has been suggested that components of an electron transport chain may be involved in this reaction. Analysis of pds1 and pds2 shows that both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene. Separate steps of the plastoquinone/tocopherol biosynthetic pathway are affected by these two mutations. The pds1 mutation affects the enzyme 4-hydroxyphenylpyruvate dioxygenase because it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and 4-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Because tocopherol-deficient mutants in the green alga Scenedesmus obliquus can synthesize carotenoids, our findings demonstrate conclusively that plastoquinone is an essential component in carotenoid synthesis. We propose a model for carotenoid synthesis in photosynthetic tissue whereby plastoquinone acts as an intermediate electron carrier between carotenoid desaturases and the photosynthetic electron transport chain.     

In vitro characterization of two different Phycomyces blakesleeanus mutants with impaired phytoene desaturation.

In vitro phytoene desaturation was investigated in two Phycomyces blakesleeanus mutants, C5 and S442, in which phytoene is accumulated instead of beta-carotene. For strain C5 but not strain S442 the phenotypic block of phytoene conversion could be overcome in vitro by the addition of Tween 40. Immunodetection of phytoene desaturase revealed in all cases the presence of a 40-kilodalton protein.     

Carotenoid biosynthesis in a Flavobacterium sp.: stereochemistry of hydrogen elimination in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin (Short Communication)

[2-(14)C,(2R)-2-(3)H(1)]- and [2-(14)C,(2S)-2-(3)H(1)]-Mevalonates were rapidly incorporated into phytoene, lycopene, rubixanthin and zeaxanthin in a Flavobacterium system obtained by disruption of the bacterial cells by shaking with glass beads. Four hydrogen atoms arising from the 2-pro-S-hydrogen atoms of mevalonate were lost in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin. The desaturation of phytoene involves trans-elimination of hydrogen in the introduction of the double bonds at C-7, C-11, C-7' and C-11'.     

Evolution of carotene desaturation: the complication of a simple pathway

In a series of desaturation reactions, the trienoic structures of phytoene and diapophytoene are extended to a maximum of 15 or 11 conjugated double bonds, respectively. After the cloning of several genes from bacteria and eukaryotes, the desaturation reactions were first analyzed in a heterologous host by functional genetic complementation. In addition, different desaturases were heterologously expressed and the reactions studied in vitro. This revealed that in archaea, non-photosynthetic prokaryotes and fungi the desaturases differ significantly from convergently evolved desaturases in cyanobacteria, Chlorobaculum (old name Chlorobium) species and eukaryotic photosynthetic organisms including plants. Detailed analysis of the desaturation reactions including the determination of the substrates converted by the enzymes, the intermediates and the products formed in the reactions revealed the bacterial all-trans desaturation pathway catalyzed by a single enzyme and the cyanobacterial/plant type poly-cis desaturation pathway which involves two closely related desaturases. This indicates that in the course of evolution of carotenogenesis from bacteria via cyanobacteria to plants, the simple situation of one enzyme for the entire reaction sequence from phytoene to all-trans lycopene changed to a more complex process. Three individual enzymes, newly acquired phytoene and ζ-carotene desaturases, as well as a carotene isomerase which is phylogenetically related to CrtI are involved. Only the CrtI-type enzymes seem to have the property to catalyze cis to trans conversion of carotenes.     

Maize w3 disrupts homogentisate solanesyl transferase (ZmHst) and reveals a plastoquinone‐9 independent path for phytoene desaturation and tocopherol accumulation in kernels

Maize white seedling 3 (w3) has been used to study carotenoid deficiency for almost 100 years, although the molecular basis of the mutation has remained unknown. Here we show that the w3 phenotype is caused by disruption of the maize gene for homogentisate solanesyl transferase (HST), which catalyzes the first and committed step in plastoquinone‐9 (PQ‐9) biosynthesis in the plastid. The resulting PQ‐9 deficiency prohibits photosynthetic electron transfer and eliminates PQ‐9 as an oxidant in the enzymatic desaturation of phytoene during carotenoid synthesis. As a result, light‐grown w3 seedlings are albino, deficient in colored carotenoids and accumulate high levels of phytoene. However, despite the absence of PQ‐9 for phytoene desaturation, dark‐grown w3 seedlings can produce abscisic acid (ABA) and homozygous w3 kernels accumulate sufficient carotenoids to generate ABA needed for seed maturation. The presence of ABA and low levels of carotenoids in w3 nulls indicates that phytoene desaturase is able to use an alternate oxidant cofactor, albeit less efficiently than PQ‐9. The observation that tocopherols and tocotrienols are modestly affected in w3 embryos and unaffected in w3 endosperm indicates that, unlike leaves, grain tissues deficient in PQ‐9 are not subject to severe photo‐oxidative stress. In addition to identifying the molecular basis for the maize w3 mutant, we: (1) show that low levels of phytoene desaturation can occur in w3 seedlings in the absence of PQ‐9; and (2) demonstrate that PQ‐9 and carotenoids are not required for vitamin E accumulation.     

Functional properties of diapophytoene and related desaturases of C(30) and C(40) carotenoid biosynthetic pathways

The desaturation reactions of C(30) carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C(40) phytoene to some extend but exhibited a high affinity to zeta-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C(40) carotenes revealed that this desaturase can also convert C(30) diapophytoene. Other homologous bacterial C(40) carotene desaturases could also utilize C(30) substrates, including one type of zeta-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C(30) carotenogenic pathway is determined by this initial enzyme which is highly homologous to C(40) phytoene synthases.     

Functional analysis of genes from Streptomyces griseus involved in the synthesis of isorenieratene,a carotenoid with aromatic end groups,revealed a novel type of carotenoid desaturase

The biosynthesis of the aromatic carotene isorenieratene is restricted to green photosynthetic bacteria and a few actinomycetes. Among them Streptomyces griseus has been used to study the genes involved in this pathway. Five genes out of seven of two adjacent operons in one cluster could be identified to be sufficient for the synthesis of isorenieratene. Stepwise deletions of these genes demonstrated their participation in phytoene synthesis, phytoene desaturation and lycopene cyclization. The novel gene crtU was assigned to encode a unique desaturase responsible for the conversion of β-carotene via β-isorenieratene to isorenieratene by a desaturation/methyltransferation mechanism. Sequence analysis of crtU revealed two conserved regions, one at the N-terminus and the other at the C-terminus of the protein which is universal to different types of carotene desaturases. In addition, the sequence comprises a motif typically found in methyltransferases. The deletion of the two remaining genes of the cluster left the carotenoid biosynthetic pathway unaffected.     

Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation.

The immutans (im) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast.     

Phytoene desaturase, CrtI, of the purple photosynthetic bacterium, Rubrivivax gelatinosus, produces both neurosporene and lycopene.

Biosynthetic pathways for carotenoids in the purple photosynthetic bacterium, Rubrivivax gelatinosus, which synthesizes spirilloxanthin in addition to spheroidene and OH-spheroidene, were investigated by means of genetic manipulation. A phytoene desaturase gene (crtI) found in the photosynthesis gene cluster of this bacterium was expressed in an Escherichia coli strain that can produce phytoene. Both neurosporene and lycopene were synthesized in the recombinant, probably by three- and four-step desaturation reactions of CrtI. A mutant of RVI: gelatinosus lacking the crtI gene produced only phytoene, indicating that this organism had no other phytoene desaturases. When the crtI deletion mutant was complemented by the three-step phytoene desaturase of Rhodobacter capsulatus, spirilloxanthin and its precursors were not synthesized, although spheroidene and OH-spheroidene were accumulated. It was concluded that neurosporene and lycopene are produced by a single phytoene desaturase in RVI: gelatinosus resulting in the synthesis of spheroidene and spirilloxanthin, and that there are no pathways for spirilloxanthin synthesis via spheroidene.     

Molecular evolution of carotenoid biosynthesis from bacteria to plants

β-Carotene and derivatives are important pigments in plant photosynthesis. They are found not only in green plants but also accumulate in archea, prokaryotes and fungi. For β -carotene biosynthesis, enzymes are necessary to catalyse the formation of phytoene, several desaturation steps and cyclization reactions. This review is focused on the molecular phylogeny of the enzymes, the genes involved and their diversity. It outlines how genes and enzymes from prokaryotes and archea were modified to give rise to the corresponding plant constituents. In the cases of phytoene synthase, a direct line of evolution can be drawn. For other carotenogenic enzymes, new genes and enzymes have been acquired at certain stages of evolution. In addition, phytoene desaturases and lycopene cyclases are examples of convergent evolution of different types of enzymes, which are structurally completely unrelated but functionally identical. Finally, several gene duplications led to homologous enzymes with different catalytic functions including those involved in the synthesis of α -carotene.     

Carotenes from Mutants of the Dinoflagellate, Crypthecodinium cohnii

SYNOPSIS.
The carotenoid compositions of 15 nitrosoguanidine-induced mutants of Crypthecodinium cohnii , a heterotrophic dinoflagellate, were determined by chromatographic and mass spectral analyses. Wild-type C. cohnii grown with irradiation of 250 W/cm² visible light at 27 C synthesizes β-carotene (33%) and γ-carotene (67%) amounting to 0.083 mg/g dry wt. There are 4 types of carotenoid-deficient mutants: (I) albinos which synthesize no C₄₀-carotonoids: (II) albinos blocked at the level of phytoene desaturation; (III) cream-colored cells which accumulate mainly §–carotene, with phytoene and/or β-zeacarotene also present; and (IV) light-orange strains which synthesize reduced amounts of β-carotene and γ-carotene.
Dark-grown wild-type cells produced 35% as much carotenoids as light-grown cells. Inhibition studies revealed that diphenylamine (3 γ) caused phytoene accumulation; nicotine at 0.9 mM blocked the final cyclization, to cause γ-carotene to accumulate in wild-type cells. Inhibition by adenine and guanine (1.5 mM) of carotenogenesis was demonstrated for the first time in any system. The effect of these purines was similar to that of diphenylamine addition: phytoene desaturation was largely inhibited.
The carotenogenic system in this dinoflagellate is similar to that of green algae and higher plants, and is under nuclear genetic control.     

A plastid terminal oxidase associated with carotenoid desaturation during chromoplast differentiation

The Arabidopsis IMMUTANS gene encodes a plastid homolog of the mitochondrial alternative oxidase, which is associated with phytoene desaturation. Upon expression in Escherichia coli, this protein confers a detectable cyanide-resistant electron transport to isolated membranes. In this assay this activity is sensitive to n-propyl-gallate, an inhibitor of the alternative oxidase. This protein appears to be a plastid terminal oxidase (PTOX) that is functionally equivalent to a quinol:oxygen oxidoreductase. This protein was immunodetected in achlorophyllous pepper (Capsicum annuum) chromoplast membranes, and a corresponding cDNA was cloned from pepper and tomato (Lycopersicum esculentum) fruits. Genomic analysis suggests the presence of a single gene in these organisms, the expression of which parallels phytoene desaturase and ζ-carotene desaturase gene expression during fruit ripening. Furthermore, this PTOX gene is impaired in the tomato ghost mutant, which accumulates phytoene in leaves and fruits. These data show that PTOX also participates in carotenoid desaturation in chromoplasts in addition to its role during early chloroplast development.     

Bansformation of tobacco with a mutated cyanobacterial phytoene desaturase gene confers resistance to bleaching herbicides

Carotenoids are constituents of the photosynthetic apparatus and essential for plant survival because of their involvement in protection of chlorophylls against photooxidation. Certain classes of herbicides are interfering with carotenoid biosynthesis leading to pigment destruction and a bleached plant phenotype. One important target site for bleaching herbicides is the enzyme phytoene desaturase catalysing the desaturation of phytoene in zeta-carotene. This enzymatic reaction can be inhibited by norflurazon or fluridone. We have transformed tobacco with a mutated cyanobacterial phytoene desaturase gene (pds) derived from the Synechococcus PCC 7942 mutant NFZ4. Characterization of the resulting transformants revealed an up to 58 fold higher norflurazon resistance in comparison to wild type controls. The tolerance for fluridone was also increased 3 fold in the transgenics. Furthermore, the transformed tobacco maintained a higher level of D1 protein of photosystem II indicating a lower susceptibility to photooxidative damage in the presence of norflurazon. In contrast, the genetic manipulation did not confer herbicide resistance against zeta-carotene desaturase inhibitors.     

Bleaching activity of 4-phenyl-3-(substituted benzylthio)-4H-1,2,4-triazoles

A variety of 4-aryl- and 4-alkyl-3-(substituted benzylthio)-4H-1,2,4-triazoles were prepared and evaluated for their bleaching activity by the lettuce seedling test. Among the series of tested compounds, 4-(3-fluorophenyl)-3-(4-trifluoromethylbenzylthio)-4H-1,2,4-triazole (39) exhibited the highest bleaching activity, causing complete bleaching symptoms at 10 microM. In the dark condition, compound 39 inhibited the formation of such carotenoids as beta-carotene, violaxanthin, neoxanthin and lutein, resulting in the formation of zeta-carotene, phytoene, phytofluene and beta-zeacarotene, which were not detected in the untreated control. Treatment by compound 39 at 50 microM resulted in the amount of accumulated zeta-carotene being seven-fold higher than that of phytoene, phytofluene and beta-zeacarotene. These results suggest that compound 39 might have interfered with desaturation, especially zeta-carotene desaturation, during carotenoid biosynthesis.     

A single five-step desaturase is involved in the carotenoid biosynthesis pathway to beta-carotene and torulene in Neurospora crassa

Phytoene desaturase Al-1 from Neurospora crassa was expressed in Escherichia coli and an active enzyme was isolated which catalyzed the stepwise introduction of up to five double bonds into the substrate phytoene. The major reaction products were 3, 4-didehydrolycopene and lycopene. Several of the desaturation intermediates, zeta-carotene, neurosporene, and lycopene, were also accepted as a substrate by Al-1. In contrast to the structurally related bacterial enzymes, the cofactor involved in the dehydrogenation reaction was NAD for Al-1. In situ competition with a neurosporene- and lycopene-converting hydratase and cyclase indicated that these enzymes can divert intermediates of the desaturation sequence. Based on the in vitro and in vivo results, the organization of the phytoene desaturase from N. crassa was proposed as an assembly of identical protein units which are responsible for the multistep reaction. However, the spatial arrangement should be loose enough to allow an exchange of individual intermediates in both directions in and out of this complex. Since gamma-carotene is not accepted as a substrate by Al-1, the formation of torulene must proceed exclusively by the cyclization of 3,4-didehydrolycopene.     

Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways

The desaturation reactions of C₃₀ carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C₄₀ phytoene to some extend but exhibited a high affinity to ζ-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C₄₀ carotenes revealed that this desaturase can also convert C₃₀ diapophytoene. Other homologous bacterial C₄₀ carotene desaturases could also utilize C₃₀ substrates, including one type of ζ-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C₃₀ carotenogenic pathway is determined by this initial enzyme which is highly homologous to C₄₀ phytoene synthases.